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SOP for Validation of Autoclave with Biological Indicators


Standard operating procedure to validate autoclave with biological indicator for sterilization efficiency.

1.0  Objective

       To validate autoclave with biological indicator for sterilization efficiency.

2.0  SCOPE

       This procedure is applicable  for Autoclave installed in microbiology area of quality control
laboratory.

3.0  Responsibility

3.1  Doing      : Tech.Assistant ( Microbiologist)/Executive
3.2  Checking : Executive/Manager    

4.0  Accountability

       Head of the Department

5.0  Procedure

5.1  Frequency  : Twice in a year

5.2  Organism : Bacillus steriothermophilus spores. (Certified Strength 106)
5.3  Perform the initial count of one strip of B.steriothermophilus spores from the lot/batch which are to be used for validation purpose.

5.4  Initial count of spores:

5.4.1  Transfer the strip aseptically into 100 ml sterile Normal Saline, keep it for 1/2 hr and give heat shock, keep the tube in water bath at 70°C-80°C for 15 minutes.
5.4.2  Shake the tube by palming or vortex it to homogenise suspension of the spores
5.4.3  Depending upon the initial count, dilute further in sterile Normal Saline
5.4.4  Take each aliquot in duplicate from different dilutions in such a way that the colony count should not come more than 300 cfu/plate.
5.4.5  Pour  the SCD agar medium which has been melted & cooled to 45oC to 50oC into the allocated plates, swirl it & let it be solidified.
5.4.6  Incubate the plates in an inverted position at 55 ± 2o C for 48-72 hours.
5.4.7  Count the colonies per plate and calculate the average number/strip using dilution factor.
5.4.8  Use the strip for validation purpose if the initial count is not more than 150 % of the label claim and not less than 75% of the label claim.
5.4.9  Record the result in Annexure-I

5.5  Autoclave validation    

5.5.1  Load the autoclave chamber with accessories and glassware.
5.5.2  Take requires nos. of strip, label it  and put inside the autoclave as describe below.
5.5.3  Place the strip in Petriplates, arrange it in autoclave as per location chart given in Annexure III & IV for double door & vertical autoclave respectively.
5.5.4  Keep one strip unexposed for POSITIVE CONTROL.
5.5.5  Run the autoclave cycle at 121oC and 15 lb pressure for 20 minutes as per S.O.P. for vertical Autoclave and as per S.O.P. for double door autoclave.
5.5.6  After cycle is over, withdraw all the strips.
5.5.7  Mark the sterile SCD (Soyabean Casein Digest) medium tubes (10ml) with location no., date and Equipment I.D. No. Transfer the strips aseptically to respective labelled SCD medium tubes.
5.5.8  Transfer one unexposed strip to a SCD medium tube and label it as POSITIVE CONTROL.
5.5.9  Keep one SCD medium tube uninoculated for NEGATIVE CONTROL.
5.5.10  Incubate all the tubes at 55oC ± 2oC for 14 days.
5.5.11  Observe the tubes daily upto 14 days for turbidity /growth in the SCD medium tubes having strips.
5.5.12  POSITIVE CONTROL should show growth within 72 hrs.

Related: New Genetically Engineered Biological Indicators

5.6  Interpretation  

5.6.1  If no growth observed in any of the SCD medium tube ,indicates satisfactory performing of equipment.
5.6.2  If growth observed in any one of the SCD medium tube, identify the growth for B.stearothermophilus performing gram staining, simultaneously compare it with POSITIVE CONTROL. In case of failure, investigate for non compliance of validation, stop use of equipment. Meanwhile rectify the cause and perform the re-validation. On compliance, equipment can be use for routine use. and repeat the cycle.
5.6.3  Maintain the validation record and affix calibration card having following criteria.
          Name  of equipment     :
          Identification No          :
          Validation frequency    :
          Validation  On             :
          Due date of Validation :
          Validation done by      :
5.7  Record the result in Annexure-II Attach the sterilization cycle record chart along with the report.

6.0  Abbreviations

       SCD= Soyabean Casein digest
       N.saline = Normal saline          
       hr.= Hour
       cfu= Colony forming unit
       B.= Bacillus 
       ml= millilitre
       %= Percentage
       oC = Degree centigrade
       U.S.P.= The United States Pharmacopoeia
       &= and
       S.O.P.= Standard operating procedure




ANNEXURE -I
MICRO BIOLOGICAL DEPARTMENT
INITIAL COUNT OF BIOLOGICAL INDICATOR
Name  of Indicator Organism  :
Claimed Initial count               :
Form of Indicator                   :
Batch /Lot No.                       :                                                              Carrier material :                                 
                         Mfg./Exp. Date :
Diluting fluid                         :  0.9% Sod.Chloride (N.Saline)
Growth medium                   :  Soyabean casein digest agar PH : _________(7.3+/-0.2) Lot No: 
                                              SCD broth                           PH : _________(7.3+/-0.2) Lot No:       
Incubation temperature         :
Incubation Period                 :
                                                                    1ml            1ml             1ml
Dilution step :  Spore'strip ---------->100 ml ----> 10ml----> 10 ml-----> 10 ml
                                                        (Dil.1)            (Dil 2)        (Dil 3)         (Dil 4 )        
Results:

Dil No.
Dil.factor
Aliquotes
plated
Cfu/plate
Mean cfu/plate
Cfu/ml
Cfu/strip
Mean cfu/strip













Actual initial count         :
Comment                      :
Sign of  Microbiologist  :
Date of Report              :
Sign. of  Q.C. Incharge :


ANNEXURE -II
MICRO BIOLOGICAL DEPARTMENT
VALIDATION OF AUTOCLAVE
Organism : Bacillus stearothermophilus  spore strip (106 cfu)                     Mfg.:                      Exp:
Label claim :
Location and No. of strip :
Batch No.of strip :
Medium :  Soyabean Casein Digest Medium (SCD)                                  Lot No:
                   pH :  __________ ( 7.3 +/- 0.2)
Incubation Temperature :  55 +/- 2oC
Sterilization :Temp: _______ oC
Cycle parameters : Hold Time :________ min.
Observation :
 Day/
 Date
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
Strip  No:














1














2














3














4














5














6














7














8














9














10














Sign
















-ve= No growth                                                                                    Negative control :_______

+ve= Growth                                                                                         Positive control :_______

Comment :
Sign. Microbiologist :
Date of Report :
Sign. of  Q.C. Incharge
ANNEXURE-I
                                                QUALITY CONTROL LABORATORY                            
VALIDATION OF AUTOCLAVE
INITIAL COUNT OF BIOLOGICAL INDICATOR

Date of Testing
: _________________________

Date of Completion
: _________
Name of Indicator Org.
Batch / Lot  No.
Mfg. / Exp. Date          
Growth medium


Incubation Temp & period 
:_________________
:_________________
:_________________
: Soyabean casein digest agar
  Lot No.:________________
  PH:____________(7.3+/-0.2)
: 53°C to 57°C for 2 to 3 Days
Autoclave Id. No.         :__________
Claimed Initial Count   :___________
Carrier Material            :__________
Diluting fluid  : 0.9% Sodium Chloride
                               


Dilution Steps:
                                                                                          1ml                 1ml                1ml                                 
                                      Carrier Material -------->100ml -------->10ml ------->10ml-------->10ml
                                                                              (dil. 1)             (dil. 2)            (dil. 3)           (dil. 4)       
Dilution
No.
Dilution
Factor
Aliquots
Plated
Cfu/plate
Mean cfu/Plate
cfu/ml
 cfu/strip
1
2











































Mean cfu / strip : ______________                                     Actual initial count : _________________


Acceptance criteria  : Initial count should NMT 150% of the label claimed & NLT 75% of the label claimed.
                                     
Analyst :                                                                                                                              Checked By :
Date     :                                                                                                                                Date           :


ANNEXURE-II
                                              QUALITY CONTROL LABORATORY
VALIDATION OF AUTOCLAVE WITH BIOLOGICAL INDICATOR

Name of Organism
B.No. / Lot No.
Mfg. / Exp. Date
Label Claim
Sterilization Temp
:
:
:
:
:
Medium Used
Autoclave Id. No.
Medium Lot No.
PH of Medium
Incubation Temp.
: Soyabean Casein Digest  medium
:
:
:
: 53oc to 57oc
Ster-hold time
Loading Time
:
Incubation Period
: 14 Days
GPT of Medium
:
Position of Spores strips : As per attached location chart

Sr. No.
Location
 Date --->
Observation (Days)
1
2
3
4
5
6
7
8
9
10
11
12
13
14













1















2















3















4















5















6















7















8















9















10















11















12















13















14















15















Sign.















+ Ve = Growth observed                                 Negative Control : _____________
- Ve = No Growth observed                            Positive Control  : ______________
Conclusion :

Analyst :                                                                                                                          Checked By :
Date      :                                                                                                                          Date             :

ANNEXURE -II
MICRO BIOLOGICAL DEPARTMENT
VALIDATION OF AUTOCLAVE
Organism : Bacillus stearothermophilus  spore strip (106 cfu)                     Mfg.:                      Exp:
Label claim :
Location and No. of strip :
Batch No.of strip :
Medium :  Soyabean Casein Digest Medium (SCD)                                  Lot No:
                   pH :  __________ ( 7.3 +/- 0.2)
Incubation Temperature :  55 +/- 2oC
Sterilization :Temp: _______ oC
Cycle parameters : Hold Time :________ min.
Observation :

Day/
Date
1st
2nd
3rd
4th
5th
6th
7th
8th
9th
10th
11th
12th
13th
14th
Strip  No:














1














2














3














4














5














6














7














8














9














10














Sign















-ve= No growth                                                                                    Negative control :_______
+ve= Growth                                                                                        Positive control :_______
Comment :

Sign. Microbiologist :
Date of Report :
Sign. of  Q.C. Incharge



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